Publication: Single molecule 3D orientation in Time and Space: A 6D dynamic study on fluorescent labeled lipid membranes.

R. Börner, N. Ehrlich, J. Hohlbein, C.G. Hübner, Journal of Fluorescence, 26, 963-975, 2016 [link]

Interactions between single molecules profoundly depend on their mutual three-dimensional orientation to each other. Recently, we demonstrated a technique that allows the orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels. As tCapture2he method is based on the detection of single photons, it additionally allows for performing fluorescence correlation spectroscopy (FCS) as well as dynamical anisotropy measurements thereby providing access to fast orientational dynamics down to the nanosecond time scale. The 3D orientation is particularly interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination at different timescales and quantifying the associated errors. The vesicles provide a well-defined spherical surface, thus, the in cooperation of lipid dyes (DiO) represents a a wide range of dipole orientations. To complement our experimental data, we performed Monte Carlo simulations of the rotational dynamics of dipoles incorporated into lipid membranes. Our study offers a comprehensive view on the dye orientation behavior in a lipid membrane with high spatiotemporal resolution representing a six-dimensional fluorescence detection approach. 

Publication: Complex coacervate core micelles with spectroscopic labels for diffusometric probing of biopolymer networks

N. Bourouina, D. de Kort, F. Hoeben, H. Janssen, H. Van As, J. Hohlbein, J. van Duynhoven, J.M. Kleijn, Langmuir, 31, 12635-12643, 2015, [link]

We present the design, TableOfContentpreparation and characterization of two types of complex coacervate core micelles (C3Ms) with cross-linked cores and spectroscopic labels, and demonstrate their use as diffusional probes to investigate the microstructure of percolating biopolymer networks. The first type consists of poly(allylamine hydrochloride) (PAH) and poly(ethylene oxide)-poly(methacrylic acid) (PEO-b-PMAA), labeled with ATTO 488 fluorescent dyes. We show that the size of these probes can be tuned by choosing the length of the PEO-PMAA chains. ATTO 488-labeled PEO113-PMAA15 micelles are very bright with 18 dye molecules incorporated into their cores. The second type is a 19F-labeled micelle, for which we used PAH and a 19F-labeled diblock copolymer tailor-made from poly(ethylene oxide) poly(acrylic acid) (mPEO79-b-PAA14). These micelles contain approximately 4 wt% of 19F and can be detected by 19F NMR. The 19F labels are placed at the end of a small spacer to allow for the necessary rotational mobility. We used these ATTO- and 19F-labeled micelles to probe the microstructures of a transient gel (xanthan gum) and a cross-linked, heterogeneous gel (kappa-carrageenan). For the transient gel, sensitive optical diffusometry methods, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching and super-resolution single nanoparticle tracking allowed us to measure the diffusion coefficient in networks with increasing density. From these measurements, we determined the diameters of the constituent xanthan fibers. In the heterogeneous kappa-carrageenan gels, bi-modal nanoparticle diffusion was observed, which is a signpost of microstructural heterogeneity of the network.

Publication: New technologies for DNA analysis – a review of the READNA Project

S. McGinn, D. Bauer, T. Brefort, L. Dong, A. El-Sagheer, A. Elsharawy, G. Evans, E. Falk-Sörqvist, M. Forster, S. Fredriksson, P. Freeman, C. Freitag, J. Fritzsche, S. Gibson, M. Gullberg, M. Gut, S. Heath, I. Heath-Brun, A.J. Heron, J. Hohlbein, R. Ke, O. Lancaster, L. Le Reste, G. Maglia, R. Marie, F. Mauger, F. Mertes, M. Mignardi, L. Moens, J. Oostmeijer, R. Out, J. Nyvold Pedersen, F. Persson, V. Picaud, D. Rotem, N. Schracke, J. Sengenes, P.F. Stähler, B. Stade, D. Stoddart, X. Teng, C.D. Veal, N. Zahra, H. Bayley, M. Beier, T. Brown, C. Dekker, B. Ekström, H. Flyvbjerg, A. Franke, S. Guenther, A.N. Kapanidis, J. Kaye, A. Kristensen, H. Lehrach, J. Mangion, S. Sauer, E. Schyns, J. Tost, J.M.L.M. van Helvoort, P.J. van der Zaag, J. O. Tegenfeldt, A.J. Brookes, K.Mir, M. Nilsson, S. Willcocks, I.G. Gut, New Biotechnology, 33, 310-330, 2016, [link]

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 4 1/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3rd and 4th generation of sequencing methods with nanopores and in situ sequencing, respectively.

Publication: Camera-based single-molecule FRET detection with improved time resolution

S. Farooq and J. Hohlbein, Physical Chemistry Chemical Physics, 17, 27862, 2015, [link], open access

The achievable time resolution of camera-based single-molecule detection is often limited by the frame rate of the camera. Especially in experiments utilizing single-molecule Förster resonance energy transfer (smFRET) to probe conformational dynamics of biomolecules, increasing the frame rate by either pixel-binning or cropping the field of view decreases the number of molecules that can be monitored simultaneously. Here, we present a generalised excitation scheme termed stroboscopic alternating-laser excitation (sALEX) that significantly improves the time resolution without sacrificing highly parallelised detection in total internal reflection fluorescence (TIRF) microscopy. In addition, we adapt a technique known from diffusion-based confocal microscopy to analyse the complex shape of FRET efficiency histograms. We apply both sALEX and dynamic probability distribution analysis (dPDA) to resolve conformational dynamics of interconverting DNA hairpins in the millisecond time range.

Publication: Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms

G.W. Evans, J. Hohlbein, T. Craggs, L. Aigrain and A.N. Kapanidis, Nucleic Acids Research, 43, 5998-6008, 2015, [link], open access

DNA polymerases maintain genoEvans2015mic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary
nucleotide. We previously employed intraprotein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase–DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening
rate in ternary complexes with complementary nucleotide was 6 s^-1, much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a  non- complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.

Publication: Studying DNA-protein interactions with single-molecule Förster resonance energy transfer

S. Farooq, C. Fijen, J. Hohlbein, Protoplasma, SPECIAL ISSUE: NEW/EMERGING TECHNIQUES IN BIOLOGICAL MICROSCOPY,  251317-332, 2014 [link]

Single-molecule Förster Protoplasma_1c-eresonance energy transfer (smFRET) has emerged as a powerful tool for elucidating biological structure and mechanisms on the molecular level. Here, we focus on applications of smFRET to study interactions between DNA and enzymes such as DNA and RNA polymerases. SmFRET, used as a nanoscopic ruler, allows for the detection and precise characterisation of dynamic and rarely occurring events, which are otherwise averaged out in ensemble-based experiments. In this review, we will highlight some recent developments that provide new means of studying complex biological systems either by combining smFRET with force-based techniques or by using data obtained from smFRET experiments as constrains for computer-aided modelling.

Publication: A Novel Parallel Nanomixer for High-Throughput Single-Molecule Fluorescence Detection

K. Mathwig, S. Schlautmann, S. G. Lemay, J. Hohlbein, A Novel Parallel Nanomixer for High-Throughput Single-Molecule Fluorescence Detection, Proceedings of the 17th International Conference on Miniaturized Systems for Chemistry and Life Science, Freiburg, Germany, Oct. 27 – 31 (2013) 1385,  [link]

This paper introduces a novel microtas2013fluidic device based on syringe-driven flow of fluorescent species through a parallel array of nanochannels, in which the geometrical confinement enables long observation times of non-immobilized molecules. Extremely low flow rates are achieved by operating the array of nanochannels in parallel with a larger microchannel. The addition of a second microfluidic inlet allows for mixing different species in a well-defined volume, enabling the study of irreversible reactions such as DNA synthesis in real-time using single-molecule fluorescence resonance energy transfer. Devices are fabricated in glass with the purpose of high-throughput single-molecule fluorescence detection.

Publication: Alternating-laser excitation: single-molecule FRET and beyond

J. Hohlbein, T.D. Craggs, T. Cordes, Chemical Society Reviews, 43, 1156-1171, 2014, [link]

The alternating-laser excitation (ALEX) EShistogramOverviewReducedscheme continues to expand the possibilities of fluorescence-based assays to study biological entities and interactions. Especially the combination of ALEX and single-molecule Förster Resonance Energy Transfer (smFRET) has been very successful as ALEX enables the sorting of fluorescently labelled species based on the number and type of fluorophores present. ALEX also provides a convenient way of accessing the correction factors necessary for determining accurate molecular distances. Here, we provide a comprehensive overview of the concept and current applications of ALEX and we explicitly discuss how to obtain fully corrected distance information across the entire FRET range. We also present new ideas for applications of ALEX which will push the limits of smFRET-based experiments in terms of temporal and spatial resolution for the study of complex biological systems.

Publication: Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion (open access)

J. Hohlbein, L. Aigrain, T.D. Craggs, O. Bermek, O. Potapova, P. Shoolizadeh, N.D.F. Grindley, C.M. Joyce, A.N. Kapanidis, Nature Communications, 4,  2131, 2013, [link], open access

The fidelity of DNA polCaptureymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.

Publication: Characterization of Dark Quencher Chromophores as Nonfluorescent Acceptors for Single-Molecule FRET

L. Le Reste, J. Hohlbein, K. Gryte, A.N. Kapanidis, Biophysical Journal, 102, 11, 2658-2668, 2012, [link]

Dark quenchers are chromophores that primarily relax from the excited state to the ground state nonradiatively (i.e., are dark). As a result, they can serve as acceptors for Förster resonance energy transfer experiments without contributing significantly to background in the donor-emission channel, even at high concentrations. Although the advantages of dark quenchers have been exploited for ensemble bioassays, no systematic single-molecule study of dark quenchers has been performed, and little is known about their photophysical properties. Here, we present the first systematic single-molecule study of dark quenchers in conjunction with fluorophores and demonstrate the use of dark quenchers for monitoring multiple interactions and distances in multichromophore systems. Specifically, using double-stranded DNA standards labeled with two fluorophores and a dark quencher (either QSY7 or QSY21), we show that the proximity of a fluorophore and dark quencher can be monitored using the stoichiometry ratio available from alternating laser excitation spectroscopy experiments, either for single molecules diffusing in solution (using a confocal fluorescence) or immobilized on surfaces (using total-internal-reflection fluorescence). The latter experiments allowed characterization of the dark-quencher photophysical properties at the single-molecule level. We also use dark-quenchers to study the affinity and kinetics of binding of DNA Polymerase I (Klenow fragment) to DNA. The measured properties are in excellent agreement with the results of ensemble assays, validating the use of dark quenchers. Because dark-quencher-labeled biomolecules can be used in total-internal-reflection fluorescence experiments at concentrations of 1 μM or more without introducing a significant background, the use of dark quenchers should permit single-molecule Förster resonance energy transfer measurements for the large number of biomolecules that participate in interactions of moderate-to-low affinity.